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1.
Ethidium bromide intercalates between the bases of native DNA, resulting in several biological anomalies. The effects of ethidium bromide on the mitochondria of cultured mouse L cells were studied. At a concentration of 1 µg ethidium bromide/ml it was observed that concentrations of cytochromes a + a3 and b decreased, a + a3 more rapidly than b. In contrast, the concentration of cytochromes c1 and c increased or remained the same as in control cells. Concomitant with the decrease of cytochromes a + a3 and b was an enlargement of the mitochondria and a reduction in the cristae. The cristae that remained were abnormally organized. After prolonged treatment with ethidium bromide a second population of small, more normally organized mitochondria was apparent. These effects of ethidium bromide could be reversed.  相似文献   

2.
Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb 561 (cytb 561). In this paper a rapid purification protocol for the cytb 561 of bean hypocotyls PM is described. An almost 200-fold increase of cytb 561 specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb 561 proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb 561 eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb 561 of higher plants, formerly suggested to be related to cytb 561 of animal chromaffin granules.Abbreviations cytb 561 cytochromeb 561 - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100  相似文献   

3.
This study describes the isolation of three HeLa cell clones after exposure of HeLa cells to ethidium bromide (EB) in culture medium for either 14 days, or 14 days plusreexposure for 30 days. All three EB-induced clones differed from the parental HeLa cell in various physical properties of their mitochondria. The ratio of mitochondrial DNA component I to component II was altered in clone HeLa-2A. In addition, the cytochrome content of the respiratory chain a + a3, b and C1 decreased, while the cytochroms c content remained unchanged. The amount of cytochromes b and c1; which were not reduced by KCN treatment, but were reduced by dithionite, increased in clone HeLa-2A. The ultrastructure of HeLa-2A cells revealed several alterations characteristic of EB treatment. Some mitochondria had enlarged profiles, a reduced number of cristae and a more lucent electron density of the matrix. Other mitochondria were tightly packed with cristae, which occasionally showed a whorled configuration. These changes were observed 4 months (20–25 passages) after the omission of EB from the medium.  相似文献   

4.
Cytochrome b561 from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent Km and maximal velocity (Vmax) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b561. The specific activity was0.7 s–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN3, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb561 produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b561.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b561. (Received May 16, 1983; Accepted September 8, 1983)  相似文献   

5.
Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a3 band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b1 was connected with their inability to reproduce under anaerobic conditions.  相似文献   

6.
Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q10 oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q10 oxidation 50%, indicating a site of action between Q10 and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q10 oxidation after acetone extraction suggests two sites for Q10 before cytochromeb.  相似文献   

7.
Cytochromec (553.7Bryopsis maxima) isolated fromB. maxima had absorption maxima at 553.7, 523.0, 417.1 and 317.5 nm in its reduced form. Isosbestic points in the reduced minus oxidized difference spectrum were located at 561, 543, 528, 511, 436, 411 and 334 nm. The purified protein exhibited a molecular weight of 10,700. The midpoint potential for the cytochromec was estimated to be 372±5 mVin vitro at pH 7.0 and 365±5 mVin vivo.In vivo 80% of the cytochromec was in the reduced form. This cytochrome was located only in chloroplasts indicating that it functions in the photosynthetic electron transport as cytochromef. Chloroplasts contained one molecule of this cytochrome per 360 molecules of chlorophyll. The magnitude of the chemically induced absorbance changes for the cytochromoesin vivo were much smaller than the light-induced absorbance change at 561 nm. It is concluded that the light-induced 561 nm absorbance change characteristic of this alga is not mainly attributable to the redox reaction of cytochromesb andf in the chloroplasts.  相似文献   

8.
Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec 1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc 1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec 1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec 1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH2, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.  相似文献   

9.
Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc 1 complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.  相似文献   

10.
F. Moreau  J.-L. Jacob  J. Dupont  C. Lance 《BBA》1975,396(1):116-124
1. An antimycin-insensitive NADH-cytochromec oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex ofHevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH.2. Twob-type cytochromes are present in the membranes. Cytochromeb563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 °K. A second cytochrome, cytochromeb561, seems to be reducible by hydrosulfite only.3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochromeP-450 could not be demonstrated.4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.  相似文献   

11.
The oxidation of cytochromeb 561 by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochromeb was calculated and expressed as a change in redox potential (E h) measured in millivolts. The oxidation of cytochromeb 561 is an energy-driven reaction affected only by the component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at GATP<–12 kcal/mole. The maximal measured value of ATP-dependent was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochromeb 561. In contrast to the ATP-induced oxidation of cytochromeb 561, cytochromeb 566 is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation ofb 561 is explained within the term of theQ cycle as a reflection of on the electron electrochemical potential. The positive electric potential of theC phase causes cytochromeb 566 to act as oxidant with respect to cytochromeb 561. In the presence of antimycin cytochromeb 561 cannot equilibrate with the quinone and undergoes oxidation, while cytochromeb 566 reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.Abbreviations used: ETPH, phosphorylating submitochondrial particles; TMPD,N 1 N 1 NN-tetramethyl-p-phenylenediamine; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; Mes, 2-(N-morpholino) ethanesulfonic acid.  相似文献   

12.
SYNOPSIS. In correlating mitochondrial structure with composition and function of the electron-transport system in Crithidia fasciculata, failure to find cytochrome oxidase in isolated mitochondria coincided with the presence of longitudinally-oriented lamellar cristae in the mitochondria in intact cells. Cytochromes b and c were detected spectrophotometrically. Respiration of intact cells and mitochondria, measured polarographically, was sensitive to 10−4 M antimycin A and 5 × 10−4 M KCN. The difficulty in detecting cytochrome oxidase and the biogenesis of mitochondria are briefly discussed.  相似文献   

13.
Summary During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb 561 (cytb 561) according to the wavelength maximum of its -band in the reduced form. More recent evidence suggests that this protein is homologous to ab-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cytb 561 is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromesb 561 probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromesb 561. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cytb 561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb 561, compares it to the animal cytb 561, and discusses the possible physiological function of these proteins in plants.Abbreviations Asc ascorbate - cyt cytochrome - DHA dehydroascorbate - E0 standard redox potential - EST expressed sequence tag - His histidine - MDA monodehydroascorbate - Met methionine - PM plasma membrane  相似文献   

14.
Exposure of HeLa and L cells to chloramphenicol causes a progressive dose-dependent decrease in cytochrome oxidase and succinate-cytochrome c reductase activities, concomitant with an increase in the amount of cytochrome c. At 2–3 days, the specific activities of the enzymes have fallen to about one-half of control values; the mitochondria appear swollen. By day 5, enzyme activities are about one-quarter of control values; the mitochondria are more swollen, with disorientation and disintegration of cristae. By day 6–8, after three generations, growth has stopped, enzyme activities are approximately the same as on day 5, and cytochrome c content has reached 170% of control value. Mitochondria show severe changes, cristae being affected more than peripheral inner membrane. The number of profiles continues to be nearly normal. After 30 days, cytochrome oxidase activity remains low but now there are mitochondria in intermediate and condensed configuration. There is a gradual accumulation in the cytoplasm of smooth membrane elements. If chloramphenicol is removed, cells recover. Ethidium bromide treatment for up to 8 days yields results virtually identical to those obtained with chloramphenicol. Cells treated with 10-4 M KCN show a decrease in cytochrome oxidase activity to about one-third of control value and an elevated amount of cytochrome c. Only a small number of mitochondria appear damaged. Autochthonous mitochondrial syntheses appear to be essential for the organization of the cristae. When cytochrome oxidase activity is impaired, a regulatory mechanism for cytochrome biosynthesis geared to mitochondrial function may be lacking, resulting in an increase in cytochrome c content.  相似文献   

15.
Electron donors such as thiosulfate, sulfite, and ascorbate have been shown to enter the respiratory chain ofT. neapolitanus at the level of cytochromec. The enzymatic oxidation of these substrates catalyzed by the cytochrome oxidase (E. C. 1.9.3.1.) ofT. neapolitanus cell-free extracts was coupled to the generation of energy which could be utilized to drive the reverse electron flow from cytochromec to pyridine nucleotides.The reduction of endogenous or added flavin by thiosulfate or ascorbate has been shown to be ATP-dependent; likewise the reduction of cytochromeb by these electron donors also required energy. The rate of ATP-driven reversal of electron transfer from cytochromec to the pyridine nucleotides was much faster compared with the rate of electron reversal catalyzed by the substrate-linked generated energy. The pathway of energy-linked reversal of electron transfer from cytochrome c to pyridine nucleotides involved cytochromeb and flavoproteins.NADH oxidation byT. neapolitanus cell-free extracts is mediated by the flavoprotein and cytochrome systems and this process also appears to be coupled with energy generation. The NADH oxidase (NADH2: cytochromec oxidoreductase) was partially inhibited by amytal or rotenone, antimycin A or HOQNO, and was relatively insensitive to cyanide or azide.This investigation was supported in part by a National Science Foundation Grant No. GB 6649 and in part by the Department of Interior, Office of Water Resources Research No. A-016-KY.  相似文献   

16.
The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 °C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number ( ) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance ( ), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 >cytochrome b5 >mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114, and ). Thus, the greatest difference in the degree of solubilization of the three cytochromes was observed with Triton X-405 ( and ). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 ( and ).  相似文献   

17.
The arrangement and function of the redox centers of the mammalianbc 1 complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH2 oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q0 center), and a Q reduction center (Q i center) situated more to the inner surface of the cristae membrane. The Q0 center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q0 center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH2, 2H+ are released to the cytosolic space and during reduction of Q, 2H+ are taken up from the matrix side, resulting in a net transport across the membrane of 2H+ per e flown from QH2 to cytochromec, the H+ being transported across the membrane as H (H+ + e) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H+ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q i center, but not via the Q0 center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.  相似文献   

18.
A cytochrome b-c1 complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c1 per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome c1 and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 ± 0.3 equivalents per cytochrome c1, while the measured content of cytochrome c1, cytochrome b561, cytochrome b565, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 ± 0.2 equivalents per cytochrome c1. It is concluded that there are no unknown redox components in the cytochrome b-c1 complex. The cytochrome b-c1 complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c1, cytochrome b561, cytochrome b566, and the Rieske iron-sulfur protein.  相似文献   

19.
Summary A nuclear gene mutant of Neurospora crassa designated cyb-3 is deficient in cytochrome b and coenzyme QH2-cytochrome c reductase. Nearly normal when grown at 25°C, the strain expresses a mutant phenotype at 38°C. Mitochondria from cybr-3 mycelium, which has undergone 3–4 mass doublings at the elevated temperature, possess 3-fold less cytochrome b, 2-fold more cytochrome, c, 5-fold less coenzyme QH2-cytochrome c reductase activity, and require 3-fold less antimycin A per milligram of protein to inhibit NADH oxidation than do wild type mitochondria. The activity of coenzyme QH2-cytochrome c reductase declines rather slowly in cultures of cyb-3 transferred to 38° C, and the in vitro thermostability of the enzyme is very similar in wild type and mutant mitochondria. Therefore, the mutation may decrease synthesis or impair integration into the membrane of cytochrome b and perhaps other proteins of the enzyme complex.Contribution No. 1294-j, Division of Biology, Agricultural Experiment Station, Kansas State University, Manhattan, Kansas.  相似文献   

20.
Differential spectrometry revealed two species for the b-type, as well as for the c-type, cytochromes in mitochondria from Agaricus bisporus Lge. The two b-type components are denoted according to their peak position in the α region at room temperature, i.e. b560 and b566. The b556 component present in all the studied higher plant mitochondria was not detected in the system. At 293 K, the c-type cytochromes exhibit a common α band with a maximum at 550 nanometers. This band is split at 77 K, with peak positions at 547 nanometers (cytochrome c) and 552 nanometers (cytochrome c1).  相似文献   

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